It is estimated that 20% of newly synthesized proteins are degraded by the proteosome due to transcriptional or translational errors [Wickner et al., Science 286:1888-1893 (1999)] and this number increases under stress conditions such as heat shock [Wickner et al., Science 286:1888-1893(1999); Ingmer et al., Res Microbiol 160:704-710(2009)]. Bacterial proteosomes also control the half-life of transcription factors and rate-limiting enzymes thereby exerting a regulatory effect on gene expression and metabolism. Thus, regulated proteolysis is critical from a quality control as well as a regulatory standpoint and loss of these intracellular proteases can have detrimental effects [Frees et al., Mol Microbiol 63:1285-1295 (2007); Jenal et al., Curr Opin Microbiol 6:163-172 (2003)]
During infection, pathogens face numerous stress conditions including nutrient deprivation, exposure to reactive oxygen species, temperature and pH changes. Loss of the caseinolytic protease (Clp) system attenuates virulence in several pathogens including B. anthracis and S. aureus [Frees et al., Mol Microbiol 63:1285-1295 (2007); Ingmer et al., Res Microbiol 160:704-710 (2009); McGillivray et al., J Innate Immun 1:494-506 (2009)] making the ClpXp protease a potential target for pharmacological intervention.
Caseinolytic proteases (Clp; EC 3.4.21.92) are endopeptidase enzymes of peptidase family S14 originally obtained from bacteria. Clp enzymes contain subunits of two types, ClpP, with peptidase activity, and ClpA or ClpX, that exhibit ATPase activity, autonomous chaperone activity and can catalyze protein unfolding
These enzymes are intracellular proteases that regulate protein quality and turnover through controlled proteolysis. Degraded proteins include damaged or non-functional proteins as well as transcriptional regulators, rate-limiting enzymes, and proteins tagged during trans-translation [Frees et al., Microbiol 63:1285-1295 (2007); Ingmer et al., Res Microbiol 160:704-710 (2009); Keiler et al., Annu Rev Microbiol 62:133-151 (2008)]. The enzymes hydrolyze proteins to small peptides in the presence of ATP and Mg2+. α-Casein is the usual test substrate. In the absence of ATP, only oligopeptides shorter than five residues are hydrolyzed.
Clp protease proteolytic core, ClpP is paired with a regulatory ATPase such as CLpA or ClpX. Clp ATPases recognize, unfold and transfer the proteins to ClpP for degradation. Orthologs of ClpXp are found in many bacterial species and are often associated with cellular stress such as heat shock, nutrient deprivation, and oxidative stress [Frees et al., Microbiol 63:1285-1295 (2007); Ingmer et al., Res Microbiol 160:704-710 (2009)]. ClpX and/or ClpP have also been implicated in virulence of several pathogens including Listeria monocytogenes, Salmonella, Staphylococcus aureus, and Streptococcus pneumoniae [Frees et al., Microbiol 63:1285-1295 (2007); Ingmer et al., Res Microbiol 160:704-710 (2009)].
The trans-translation mechanism is a key component of multiple quality control pathways in bacteria that ensure proteins are synthesized with high fidelity in spite of challenges such as transcription errors, mRNA damage, and translational frameshifting. trans-Translation is performed by a ribonucleoprotein complex composed of tmRNA, a specialized RNA with properties of both a tRNA and an mRNA, and the small protein SmpB. tmRNA-SmpB interacts with translational complexes stalled at the 3′ end of an mRNA to release the stalled ribosomes and target the nascent polypeptides and mRNAs for degradation. In addition to quality control pathways, some genetic regulatory circuits use trans-translation to control gene expression. Diverse bacteria require trans-translation when they execute large changes in their genetic programs, including responding to stress, pathogenesis, and differentiation.
The compound F2, below, was identified as
part of a high-throughput screen for inhibitors of the protein-tagging and trans-translation degradation pathway in E. coli. F2 has been found to inhibit the activity of ClpXp protease of bacterial cells with minimal host cell cytotoxicity [McGillivray et al., J Innate Immun 1:494-506 (2009)]. Although it is unclear exactly how F2 inhibits the ClpXp protease, the data indicate that inclusion of F2 decreases the proteolysis of ClpXp substrates in vivo. Similar to a genetic loss of ClpX, co-treatment of B. anthracis with F2 increased susceptibility of the bacteria to cathelicidin antimicrobial peptides. A similar effect was seen with both methicillin-susceptible and methicillin-resistant strains of S. aureus suggesting ClpXp also plays a role in cathelicidin resistance in S. aureus. The McGillivray et al. paper is the first study to show that inhibition of ClpXp could result in a synergistic interaction with innate immune defenses.
In that paper, McGillivray and co-workers demonstrated that ClpX is critical for the pathogenesis of Bacillus anthracis, a Gram-positive bacterium that is the causative agent of anthrax [J Innate Immun 1:494-506 (2009)]. Loss of ClpX increased susceptibility to innate host defenses including cationic antimicrobial peptides and severely attenuated B. anthracis virulence even in the fully pathogenic Ames strain. Ibid.
Although McGillivray et al. focused upon cathelicidins in that study, they had previously shown loss of ClpX renders B. anthracis more sensitive to other antimicrobial proteins, including defensins and lysozyme [McGillivray et al., J Innate Immun 1:494-506 (2009)]. Therefore, pharmacological inhibition of ClpXp may increase susceptibility to multiple host defenses.
F2 also sensitizes B. anthracis and S. aureus to antibiotics such as penicillin and daptomycin, although the synergistic effect between F2 and antibiotics was more pronounced in B. anthracis. That enhancement may reflect the assay conditions or it may indicate that the extent to which ClpXp influences susceptibility to cell-envelope antibiotics differs among species.
Evidence of a connection between Clp proteases and cell-wall acting antibiotics has been seen in other bacterial species. Loss of ClpP in Streptococcus mutans rendered the bacteria more susceptible to the cell wall acting antibiotics bacitracin, polymyxin B, and vancomycin, although no effect was seen with non-cell wall acting antibiotics [Chattoraj et al., J Bacteriol 192:1312-1323 (2010)].
In Mycobacterium tuberculosis, loss of the ClpCP protease, another ATPase chaperone, resulted in increased susceptibility to cell wall stress induced by vancomycin or SDS [Barik et al., Mol Microbiol 75:592-606 (2010)]. Daptomycin is believed to function by membrane depolarization but a recent study demonstrated that it also induces the cell wall stress regulon including Clp family members in S. P aureus [Muthaiyan et al., Antimicrob Agents Chemother 52:980-990 (2008)].
The importance of the ClpXp system is further highlighted by those workers' observation that inhibition increased susceptibility to daptomycin in otherwise non-susceptible strains. However, this suppression of resistance was only partial, indicating that non-Clp-dependent effects probably also contribute to daptomycin resistance. It is likely that loss of ClpXp has pleiotropic effects on the bacterial cell because the Clp protease regulates a wide range of genes [Michel et al., J Bacteriol 188:5783-5796 (2006); Robertson et al., J Bacteriol 184:3508-3520 (2002)].
Cell wall active agents are believed to increase damaged or mis-folded proteins and result in induction of genes involved in protein turnover such as chaperones and proteases [[Muthaiyan et al., Antimicrob Agents Chemother 52:980-990 (2008); Utaida et al., Microbiology 149:2719-2732 (2003)]. Loss of ClpXp could hamper this response. The ClpXp protease may also be regulating critical components of the cell wall. In E. coli, ClpXp can degrade FtsZ, a major cytoskeletal protein that is implicated in cell division and cell wall synthesis [Camberg et al., Proc Natl Acad Sci USA 106:10614-10619 (2009)]. In B. subtilis, MurAA, an enzyme important in peptidoglycan formation, is degraded by ClpCP [Kock et al., Mol Microbiol 51:1087-1102 (2004)].
It is also possible that cell charge is affected by loss of ClpXp. Daptomycin is an anionic compound that associates with calcium to form a cationic complex similar to an antimicrobial peptide. Resistance to daptomycin and cationic antimicrobial peptides has been linked to mutations in mprF (lysine addition to cell membrane phosphatidyl glycerol) and the dltABCD operon (alanylation of cell wall teichoic acids) that result in increased net positive surface charge in both S. aureus and B. anthracis [Fisher et al., J Bacteriol 188:1301-1309 (2006); Kraus et al., Curr Top Microbiol Immunol 306:231-250 (2006); Samant et al., J Bacteriol 191:1311-1319 (2009); Yang et al., Antimicrob Agents Chemother 53:2636-2637 (2009)].
Consistent with this hypothesis, increasing daptomycin resistance in S. aureus was accompanied by increased resistance to cationic antimicrobial peptides such as alpha-defensin HNP-1 and platelet microbicidal proteins [Jones et al., Antimicrob Agents Chemother 52:269-278 (2008)]. The Clp protease could be regulating expression either directly or indirectly of the mprF or the dltABCD operon although this has not yet been demonstrated.
The ClpXp protease is a promising target for pharmacological intervention. Inclusion of F2 increased the effectiveness of bacterial killing by human whole blood indicating this compound can augment innate immune defenses. This therapeutic effect may be magnified at tissue sites of infection where high levels of antimicrobial peptides are produced by cells such as keratinocytes, and in patients receiving concurrent antibiotic therapy with cell wall active agents for which ClpXp inhibition also provides synergism.
Although McGillivray et al. focused on inhibition of ClpXp, uncontrolled activation of ClpP through a new class of antibiotics, acyl depsipeptides, also has lethal consequences for several bacterial species tested [Brotz-Oesterhelt et al., Nat Med 11:1082-1087 (2005)]. However, as was seen with acyl depsipeptides [Brotz-Oesterhelt et al., Nat Med 11:1082-1087 (2005)] and other antimicrobial compounds, potential for bacterial resistance to F2 exists. The mechanism behind this resistance is at this point unclear, but it may necessitate that F2 or another pharmacological inhibitor of ClpXp be used in a combination rather than single therapy to limit resistance. Nevertheless, the use of Clp inhibitors can be predicted to contribute to antimicrobial activity on multiple levels, through increased susceptibility to innate immune defenses and decreased resistance to traditional antibiotics, potentially increasing therapeutic effectiveness.
An alternative strategy in antimicrobial therapy is to target and inactivate bacterial virulence factors rather than directly targeting growth or survival in the manner of traditional antibiotics [Cegelski et al., Nat Rev Microbiol 6:17-27 (2008)]. Inhibition of virulence factors involved in disease progression should enhance the ability of the host immune system to clear the pathogen. The ClpXp protease is a promising target for pharmacological inhibition due to its conserved nature and its role in the virulence of a wide-variety of pathogens.
A present inventor and colleagues have identified several inhibitors of ClpXp using a screening system devised in E. coli [Cheng et al., Protein Sci 16:1535-1542 (2007)]. The ClpXp protease of the Gram positive B. anthracis Sterne and the Gram positive human pathogen, Staphylococcus aureus were targeted using the F2 inhibitor. It was found that F2 renders both B. anthracis Sterne and drug-resistant strains of S. aureus more susceptible to host antimicrobial peptides as well as antibiotics that target the bacterial cell envelope including the cell wall and/or cell membrane.